Plastids are ideal for genetic engineering because they offer a number of attractive advantages, including high-level transgene expression (Daniell et al., 2002), multi-gene engineering in a single transformation event (DeCosa et al., 2001; Ruiz et al., 2003; Daniell & Dhingra, 2002), transgene containment via maternal inheritance (Daniell 2002), lack of gene silencing (Lee et al., 2003; DeCosa et al., 2001), position effect due to site specific transgene integration (Daniell et al., 2002) and pleiotropic effects (Daniell et al., 2001; Lee et al., 2003). Chloroplast genetic engineering is most suitable for hyper-expression of vaccine antigens and production of valuable therapeutic proteins. Ever since we demonstrated expression of human-elastin derived polymers for various biomedical applications (Guda et al., 2000), we have extended this approach to express vaccine antigens for Cholera and Anthrax (Daniell et al., 2001, Daniell 2003), monoclonal antibodies (Daniell et al., 2001) and human therapeutic proteins, including Human Serum Albumin (Fernandez et al., 2003), Magainin (DeGray et al., 2001), Interferon (Daniell 2003) and Insulin-like Growth Factor (Daniell, 2003). Several other laboratories have expressed Human Somatotropin and Interferon-GUS fusion proteins to improve stability (Reddy et al., 2003) and tetanus vaccine antigens (Tregoning et al., 2003) in transgenic chloroplasts. Without any exception, all of these therapeutic proteins have been expressed in transgenic tobacco chloroplasts, meeting the zero-tolerance of food crops for plant-derived pharmaceuticals advocated by various environmental groups.
However, there is an urgent need for oral delivery of therapeutic proteins and vaccine antigens to dramatically reduce their production, purification, storage and transportation costs and minimize complications associated with intravenous delivery. Carrot (Daucus carota L.) is one of the most important vegetables used worldwide for human and animal consumption, due to its excellent source of sugars, vitamins A and C, and fiber in the diet. The carrot plant is biennial, completing its life cycle in two years. In the first year the plant produces the fleshy taproot, which is edible. If left in the ground, plants flower in the second year after passing through a cold season (Yan, W. & Hunt, L. A Reanalysis of Vernalization Data of Wheat and Carrot, Annals of Botany 84, 615-619 (1999)). In addition, chloroplast genomes in the cultivated carrot crop are transmitted strictly through maternal inheritance (Vivek et al. 1999). Thus, carrot is environmentally safe and is doubly protected against transgene flow via pollen and seeds to achieve zero-tolerance on transgene flow advocated for food crops. Carrot somatic embryos are single cell derived and multiply through recurrent embryogenesis; this provides a uniform source of cell culture, which is one of the essential requirements for producing therapeutic proteins (homogeneous single source of origin). Carrot cells divide rapidly and a large biomass is produced using bioreactors. Cultured carrot cells are edible and could be used directly to deliver precise doses of vaccine antigens or biopharmaceuticals. When delivered via edible carrots, there is no need to cook and this would preserve the structural integrity of therapeutic proteins during consumption. Viable for long duration on culture medium, encapsulated embryos are used as synthetic seeds for cryopreservation and controlled germination (Tessereau, H., B. Florin, M. C. Meschine, C. Thierry and V. Pétiard, 1994). Thus, transgenic carrot with enhanced medicinal or nutritional value can play a vital role in improving human or animal health.
However, there is a need for more efficient plastid transformation including an efficient process to transform important crop species which allows for the regeneration of transplastomic plants via somatic embryogenesis.
Table 1 shows an exemplary list of the development of transgene expression in chloroplasts.
TABLE 1Transgene Expression in ChloroplastsAgronomictraitsGenePromoter5′/3′ Regulatory elementsReferenceInsect resistanceCry1A(c)PrrnrbcL/Trps16Mc Bride et al.1995HerbicideCP4Prrnggagg/TpsbADaniell et al.resistance(petunia)1998Insect resistanceCry2Aa2Prrnggagg (native)/TpsbAKota et al. 1999HerbicideCP4PrrnrbcL or T7 gene 10/Ye at al 2001resistance(bacterialTrps16orsynthetic)Insect resistanceCry2Aa2PrrnNative 5′UTRs/TpsbADeCosa et al.operon2001DiseaseMSI-99Prrnggagg/TpsbADeGray et al.resistance2001Salt and droughttpsPrrnggagg/TpsbALee et al. 2003tolerancePhytoremediationmerAa/merPrrnggagga,b/TpsbARuiz et al. 2003Bb5′/3′Biopharma-regulatory% tspceutical proteinsGenePromoterelementsexpressionReferenceProtein basedEG121PrrnT7gene10/Not testedGuda et al. 2000polymerTpsbAHumanhSTPrrna,T7gene10a or7.0%a andStaub et al. 2000somatotropinPpsbAbpsbAb/Trps161.0%bCholera toxinctxBPrrnggagg/TpsbA4%Daniell et al.2002Tetanus toxinTetCPrrnT7 gene 10a,25%a,Tregoning et al.(bacterialatpBb/TrbcL10%b2003andsynthetic)Human SerumhsaPrrna,ggagga, psbAb/0.02%a,Fernandez-SanAlbuminPpsbAbTpsbA11.1%bMilan et al. 2003Interferon alpha 5INFα5PrrnPpsbA/TpsbANDTorresInterferon alphaINFα2BPrrnPpsbA/TpsbA19%Falconer2BInterferon gammaifn-gPpsbAPpsbA/TpsbA6%Leelavathi andReddy, 2003MonoclonalPrrnggagg/TpsbANDDaniell et al.antibodies(photosynthesis)Insulin like growthIgf-1PrrnPpsbA/TpsbA33%Ruiz GfactorAnthrax protectivePagPrrnPpsbA/TpsbA4-5%WatsonantigenPlague vaccineCaF1~LcrVPrrnPpsbA/TpsbA4.6%Singleton